title: Monitoring voltage-dependent charge displacement of Shaker B-IR K+ ion channels using radio frequency interrogation.
abstract: Here we introduce a new technique that probes voltage-dependent charge displacements of excitable membrane-bound proteins using extracellularly applied radio frequency (RF, 500 kHz) electric fields. Xenopus oocytes were used as a model cell for these experiments, and were injected with cRNA encoding Shaker B-IR (ShB-IR) K(+) ion channels to express large densities of this protein in the oocyte membranes. Two-electrode voltage clamp (TEVC) was applied to command whole-cell membrane potential and to measure channel-dependent membrane currents. Simultaneously, RF electric fields were applied to perturb the membrane potential about the TEVC level and to measure voltage-dependent RF displacement currents. ShB-IR expressing oocytes showed significantly larger changes in RF displacement currents upon membrane depolarization than control oocytes. Voltage-dependent changes in RF displacement currents further increased in ShB-IR expressing oocytes after ∼120 µM Cu(2+) addition to the external bath. Cu(2+) is known to bind to the ShB-IR ion channel and inhibit Shaker K(+) conductance, indicating that changes in the RF displacement current reported here were associated with RF vibration of the Cu(2+)-linked mobile domain of the ShB-IR protein. Results demonstrate the use of extracellular RF electrodes to interrogate voltage-dependent movement of charged mobile protein domains--capabilities that might enable detection of small changes in charge distribution associated with integral membrane protein conformation and/or drug-protein interactions.
authors.affiliation: Department of Bioengineering, University of Utah, Salt Lake City, Utah, United States of America. sameera_dharia@yahoo.com
authors.forename: Sameera
authors.initials: S
authors.lastname: Dharia
authors.forename: Richard D
authors.initials: RD
authors.lastname: Rabbitt
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D003300:Copper / Q000494:pharmacology; D017097:Electric Impedance; D055724:Electrophysiological Phenomena / Q000187:drug effects / Q000502:physiology; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D008954:Models, Biological; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000379:methods*; D011846:Radio Waves; D051637:Shaker Superfamily of Potassium Channels / Q000378:metabolism / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: C076413:Shaker B potassium channel polypeptide; D051637:Shaker Superfamily of Potassium Channels; D003300:Copper